Mutation Analysis and Genotyping

Improve the detection range of your assays, boost the discriminatory power of your analyses, and expand the spectrum of genetic variation that you can examine. Pyrosequencing can enhance genetic analyses by delivering explicit and quantitative sequence data. Furthermore, independent studies have demonstrated that Pyrosequencing shows full concordance with Sanger sequencing, but is more cost-effective and less labor-intensive.

Many mutation types are important in fields such as epidemiology, pharmacogenetics, and animal husbandry, and new markers are continuously being discovered. As a result, a variety of analysis methods are used to detect genetic variability. This can complicate comparison of results obtained by different methods and different researchers. Ideally, a single technology could be adapted for all applications to standardize results. Pyrosequencing offers precisely that versatility. Because of the flexibility of primer placement in Pyrosequencing reactions, virtually all genetic markers — those currently in use as well as those yet to be identified — can be assayed. Alleles of variable loci are accurately quantified, and heterozygosity is easily resolved (see figures Analysis of a tri-allelic SNP and Quantitative mutation analysis). In addition, because Pyrosequencing delivers sequence information, various types of genetic variation can be evaluated — insertion-deletions, single nucleotide polymorphisms, single tandem repeats, and variable gene copy number — and it is possible to assay several contiguous sequence variants in a single run.

Analysis of complex mutations

Regardless of the marker or mutation being analyzed, preparation of templates for Pyrosequencing and the subsequent analysis of the resulting sequence information are quick and easy, saving time and valuable resources. However, the strength of Pyrosequencing for genetic testing lies in the elegance of its output. Because results are simply the true sequence of the DNA in a sample, the user can examine multiple mutation sites within a specified region, and even multiple variation types, all in the same run (see figure Two mutation types quantified in a single Pyrosequencing reaction). The improved chemistry of the PyroMark Q24 Advanced system also enables analysis of multiple mutations over long sequences (see figure Quantitative mutation analysis in long sequence runs).

Furthermore, the straightforward results are easily interpreted. Pyrosequencing enables de novo sequencing which, coupled with the built-in control afforded by the sequence surrounding the variable site, is a guaranteed way of validating newly identified markers. The high throughput facilitates rapid compilation of the population data needed to establish reference databases for these markers. This feature makes Pyrosequencing a powerful and versatile tool for the development of pharmacogenetic markers (1).